This invention relates to biochemical assays for a wide class of free fatty acids, retinol and wherein the analyte is caused to react with a specific binding fluorescently modified, small molecular weight protein, and thereby causes a detectable fluorescence signal. A one-step assay for free fatty acids, either in whole blood, serum, food preparations, or various laboratory conditions using a suitably altered fatty-acid-binding protein (FABP) is provided. To date, fourteen different probes for detecting and measuring free fatty acids have been constructed using the principles taught hereinafter.
Long chain free fatty acids (FFA) with acyl chains&gt;16 carbons are quantitatively the most important physiological energy source. While ubiquitous and essential for normal physiological function, FFA are also potent modulators of cellular activity, Karnovsky, M. J., Kleinfeld, A. M., Hoover, R. L. and Klausner, R. D. J. Cell Biol. 49:1-6, 1982; Richieri, G. and Kleinfeld, A. M., J. Immunol. 145:1024-1077, 1990. There are, in fact, numerous indications that FFA levels uniquely reflect various states of health and disease. Variations in total fatty acid (FA) levels have been reported in a number of pathologies including AIDS, ischemia, inflammation, diabetes, immune dysfunction, and cancer. Brown, R. E., Steele, R. W., Marmer, D. J., Hudson, J. L. and Brewster, M. A. J. Immunol. 131:1011, 1983; Hochachka, P.W. Science 231:234, 1986; Levy, J.A. in Basic and Clinical Immunology, D. P. Stites. J. D. Stobo, H. H. Fudenberg, and J. V. Wells, eds., Lange Medical Publications, Los Altos, CA, pp. 293-301, 1984; Reaven, G. M., Hollenbeck, C. Jeng, C. Y, Wu, M. S. and Chen, Y-D.I. Diabetes 7:1020, 1988; Tsuchiya, H., Hayashi, T., Sato, M., Tatsumi, M. and Takagi, N. J. Chromatogr. 309:43, 1984.
In specific instances, the concentration of FFA may be of significant importance in the diagnosis or treatment of disease or in studying the underlying biochemical or immunochemical causes or effects of disease. For example: FFA are believed to be important factors in the cause of ventricular arrhythmias during acute myocardial infarction, Makiguchi M, Hokkaido Igaku Zasshi (JAPAN) Jul 1988, 63 (4) p 624-34. Significant differences in free fatty acids from normal levels in AIDS patients may be implicated in the pathophysiology of AIDS and could represent a good index of diagnosis and prognosis, Christeff, N.; Michon, C.; Goertz, G.; Hassid, J.; Matheron, S.; Girard, P.M.; Coulaud, J. P.; Nunez, E. A. et. al., EUR. J. CANCER CLIN. ONCOL.; 24(7), pp. 1179-1183 1988. Ambient plasma free fatty acid concentrations in non-insulin-dependent diabetes mellitus may be indicative of insulin resistance, Fraze, E.; Donner, C. C.; Swislocki, A.L.M.; Chiou, Y.-A.M.; Chen, Y.-D.I.; Reaven, G. M., J. CLIN. ENDOCRINOL. METAB.; 61(5), pp. 807-811 1985. Fatty acids have been implicated in the pathogenesis of thromboatherosclerosis, Tavella, M.; Mercuri, 0.; de Tomas, M.E., NUTR. RES.; 5(4), pp. 355-365 1985. Depression of serum calcium may result from increased plasma free fatty acids, Warshaw, A. L.; Lee, K.-H.; Napier, T. W.; Fournier, P. O.; Duchainey, D.; Axelrod, L., GASTROENTEROLOGY; 89(4), pp. 814-820 1985.
Elevated levels of FA have been found in human cancer patients and murine models (Ligaspi, A., Jeevanandam, M., Starnes, H. F., & Brennan, M. F., Metabolism 36:958, 1987; Iguchi, T., Takasugi, N., Nishimura, N., & Kusunoki, S. Cancer Res. 49:821, 1989; Brown, R. E., Steele, R. W., Marmer, D. J., Hudson, J. L., & Brewster, M. A. J. Immunol. 131:1001, 1983) and these elevated levels were shown to result in immunological deficiencies (Brown, R. E., Steele, R. W., Marmer, D. J., Hudson, J. L., & Brewster, M. A. J. Immunol. 131:1001,1983).
In addition to their importance in disease, the measurement of FFA levels has important applications in a wide variety of biochemical, biophysical, cell biologic, and physiological research. These include studies of FFA transport (Storch, J. and Kleinfeld, A.M., Biochemistry 25:1717, 1986; Potter, B. J., Sorrentino, D. and Berk, P. D., Ann. Rev. Nutr. 9:253, 1989), inter and intra-cellular signalling (Kim, D., Lewis, D. L., Graziadel, L., Heer, E. J., Bar-Sagi, D. and Clapham, D. E., Nature 337:557, 1989), and membrane structural perturbation (Karnovsky, M. J., Kleinfeld, A. M., Hoover, R. L., and Klausher, R. D. J. Cell Biol. 49:1, 1982.) The study of Storch et. al., in which the present inventor was a major participant, was designed to use special (synthetic-fluorescent) FFA to probe the structure of the protein, not to determine the aqueous phase concentration of FFA. The technique described by Storch et. al. cannot be used to measure the concentration of natural FFA. FFA found in serum (natural FFA) have-no fluorescent or any other groups that can be used for detection so the only way to detect them is to make the protein fluorescent. In this invention, it is the protein that is fluorescent, as it must be for the present method to work. Storch et. al. measured the binding to the fatty binding protein of synthetic (fluorescent) FA that have an anthracene group covalently attached through an ester linkage (the AOFFA). Storch et. al. did not determine the aqueous phase (or even protein bound) concentration of these AOFFA because they could not. The method that storch et. al. employed does not allow quantitation of the AOFFA bound to the protein. To do this would require a determination of the absolute quantum yield of the AOFFA within the protein binding site and this was not done. The fluorescence of the AOFFA does not shift when AOFFA moves from water to protein. In other words there is only one wavelength (band) of emission whether there is binding or not. The shift in wavelength that the present inventor discovered and discloses herein occurs for the probes in the present invention. It is this shift in emission wavelength that is particularly important to quantitation of the absolute FFA concentration and which is important in the invention described in this patent.
While vital information about both normal and pathological physiology would accrue from the measurement of plasma levels of FFA, there have been two essential barriers to obtaining this information. First, no method has previous existed for measuring the aqueous phase concentration of unbound FA (FFA). Direct measurements of FFA have I 0 not been possible previously because their low aqueous phase solubility causes long chain FA to adhere to virtually all surfaces and therefore monomer concentrations, in aqueous solutions, cannot be determined by physical separation. The un-esterified and unbound (as opposed to bound to serum albumin) FFA in the aqueous phase is, however, the active form of the FA in both normal and pathologic states. Second, although the concentration of unbound FFA can be estimated, this can be done only with considerable effort. The unbound FFA concentration is estimated from the ratio of total serum FA to total serum albumin. Once the total FA and total albumin have been measured, the unbound FFA concentration is calculated using the FA-albumin association coefficients (.about.8 different sites/albumin molecule) determined from measurements of FFA partition between an albumin-water phase and heptane, Ashbrook, J. D., Spector, A. A., Santos, E. C. and Fletcher, J. E. J. Biol. Chem. 250:2233, 1975, Richieri, G. V., Anel, A., and Kleinfeld, A. M., Biochemistry 32; 7574, 1993. Typical values calculated in this fashion yield concentrations in the range of 2 to 200 nM. Even this estimate, however, can only be obtained with considerable effort since the determination of both total FA and total albumin concentrations requires several very time-consuming and expensive steps.
Determination of total FA and albumin first requires the separation of plasma and cellular components in whole blood. This is done by centrifugation and decantation of the upper (plasma) phase. A colorimetric or ELISA assay can be used for determining the plasma concentration of albumin. Several approaches have been used to determine total FA. In the most common, the lipid fraction must first be extracted from total plasma using, essentially, the method of Folch et. al., Folch, J., Lees, M. and Stanley, G. H. S. J. Biol. Chem. 226:497, 1957. This method involves suspension of a quantity of plasma fluid in a solution of chloroform:methanol, centrifugation of this mixture and decantation of the supernatant, re-extraction of the residue with methanol:chloroform:water, centrifugation of this second mixture followed by decantation of the second supernatant and combination with the first. Chloroform is added to the combined supernatants, a twophase mixture is produced by centrifugation, and the lower chloroform phase which contains the lipid is saved. At this stage most previous workers have concentrated the chloroform phase, derivatized the fatty acids to methyl esters and then performed chromatography to quantirate the various components. Baty, J. D. and Pazouki, S. J. Chromat. 395:403, 1987. It is possible, however, to determine the total FA content of a heptane extract by adding a chloroform solution containing a divalent cation such as 63Ni to the heptane extract. A two phase system is produced in which the upper phase contains complexes (probably micelies) of 63Ni complexed with the anionic FFA and the total FFA concentration can be determined from the 63Ni activity, Ho, R.J. Anal. Blochem. 36: 105, 1970. A variation on these approaches involves the direct derivatization of FA while in plasma, using visible-UV FFA reactive reagents (Miwa, H., Yamamoto, M., Nishida, T., Nunoi, K. and Kikuchi, M. J. Chromat. 416:237, 1987. The derivatized FFA complex is then extracted from plasma using organic solvents and FFA are then assayed using HPLC.
Another method for estimating lipid component is described by Imamura Shigeyuki, et. al., U.S. Pat. No. 4,491,631, wherein an enzyme having enoyl-coA hydratase activity, 3-hydroxyacyl-coA dehydrogenase activity and 3-ketoacyl-coA thiolase activity, all in the same enzyme, is produced by culturing the microorganism strain Pseudomonas fragi b-0771 FERM-p no. 5701, and isolating the enzyme thus produced from the culture medium. The enzyme is useful in an assay method for a fatty acid component in a sample, which fatty acid is originally present in the sample or is liberated from a fatty acid ester in the sample. The assay is carried out by converting the fatty acid to acyl-coA, converting the thus-produced acyl-coA to dehydroacyl-coA; converting the thus-produced dehydroacyl-coA to hydroxyacyl-coA, converting the thus-produced hydroxyacyl-coA to ketoacyl-coA, converting the thus-produced ketoacyl-coA to acyl-coA and measuring the detectable changes in the reaction mixture.
These assay methods generally are complicated and time consuming, and are not readily carried out in any but a well-equipped research laboratory. Moreover they are determinations of the total FA not FFA; these total FA values can only be used to estimate FFA. Thus, there is a critical need for an assay for free fatty acids (FFA) in any aqueous solution including plasma, serum and blood.